Although significant knowledge has recently been gained on the incorporation of selenium into selenoproteins, most of the emphasis of this work has been placed on enzymes that carry selenium in the form of selenocysteine. A handful of enzymes have been characterized in which selenium is present and required for activity but is not inserted during translation of the mRNA and therefore is not present as selenocysteine. Two such enzymes are nicotinic acid hydroxylase (NAH) and xanthine dehydrogenase (XDH) from Clostridium. Selenium present in NAH is somewhat labile and can be removed by treatment with chaotropic agents, such as sodium dodecyl sulfate. In order to better understand the nature of selenium and the incorporation of selenium, XDH from Clostridium purinolyticum was purified to homogeneity. XDH was found to consist of three subunits (based on SDS-PAGE) and to be labeled with selenium (75-Se). This selenium was labile and presumably is not in the form of selenocysteine. During the isolation of this enzyme, another selenium-containing hydroxylase was isolated and termed purine hydroxylase (PH). PH consisted of four subunits (based on denaturing gel electrophoresis) and utilized purine and hypoxanthine as substrates. Selenium in PH was also labile, and thus represents yet another non-selenocysteine selenium-requiring enzyme to be characterized and to be used in a comparative analysis of these unique enzymes. Now that a good purification scheme has been determined for the isolation of both XDH and PH from C. purinolyticum, these enzymes will be investigated in more detail. These investigations will focus on the identification of cofactors and metals present, as well as study of the active sites for enzyme reaction and electron transfer using methods such as electron paramagnetic resonance (EPR) and x-ray absorption spectroscopy (XAS). The focus of the investigation will revolve around determining the moiety of selenium present in the active site and its relationship to other cofactors (e.g. molybdenum cofactor). Once more knowledge is gained into the properties of selenium in these enzymes, the mechanism by which the cell incorporates selenium into this active site will be investigated. This work may uncover a new delivery protein(s) or selenium donor molecule required for XDH and PH and possibly other molybdenum hydroxylases, and perhaps shed light on the delivery of selenium in the biosynthesis of selenocysteine.